Dissecting the genetic determinants of bacterial DNA degradation by bacteriophage T5

Upon infection of Escherichia coli , the virulent bacteriophage T5 employs a unique two-step mechanism to transfer its 122-kb genome: only 8% of the DNA are initially transferred, allowing expression of pre-early genes that alter host functions before the remaining DNA is delivered. Early infection triggers rapid host DNA degradation and nucleotide catabolism, processes partly controlled by the pre-early genes encoding the predicted metallo-phosphatase A1 and the dNMP phosphatase Dmp. However, the functions of most of the 17 proteins encoded by the first-step transfer DNA (FST-DNA) remain unknown.

Using reverse genetics, we engineered several T5 mutants carrying deletions in the FST-DNA. One of them carries only four pre-early genes ( A1 , A2 , hdi and 009 ), demonstrating that thirteen of the 17 predicted genes are dispensable under laboratory conditions. Mutant characterization showed that only A1 and the predicted DNA-binding protein gene A2 are essential for productive infection, while dmp enhances phage virulence. Notably, seven pre-early genes ( dmp , A1 , hdi , hegG , 011 , 013 and 015 ) proved toxic when expressed in E. coli without other viral factors, causing severe morphological changes or nucleoid disorganization, while one gene compromised membrane integrity. The essential gene A1 , which is conserved among all viruses in the Demerecviridae family, emerged as the primary driver of host genome degradation, both essential and sufficient for chromosomal DNA digest in vivo .

These findings advance our understanding of how T5 manipulates its host during the critical early stages of infection, offering new insights into phage-host interactions and the molecular strategies viruses use to subvert bacterial cells.